转基因产品中bar和pat基因的SYBR Green I-PCR检测方法研究(英文)

SYBRGreenI是一种在实时定量PCR检测中广泛使用的DNA结合染料。PCR结果可以通过溶解曲线分析来鉴定而不再需要使用传统的琼脂糖凝胶电泳来鉴定,因为PCR特定扩增产物的溶解温度的特异性是类似于琼脂糖凝胶电泳中的扩增条带的特异性。建立了基于扩增产物溶解曲线分析的二重实时定量PCR对转bar和pat基因的作物进行检测的方法。获得了bar和pat基因的特异性扩增产物和他们的特定溶解温度,并且通过几组不同的二重PCR对他们的扩增产物进行了溶解曲线鉴定。还对影响溶解曲线分析的一些因素进行了研究和讨论。结果表明基于扩增产物的溶解温度建立的二重PCR检测方法是可行的,该方法对转bar和pat基因的作物可达到0.9%的检测限。

SYBR Green I Based PCR for Detection of the bar and pat Genes in Genetically Modifed Organisms

SYBR Green I is widely used in real-time PCR applications as an intercalating dye. The use of melting curve analysiseliminates the necessity for agarose gel electrophoresis because the melting temperature (Tm) of the specific amplicon isanalogous to the detection of an electrophoretic band. In this paper we reported the development of a duplex real-time PCR suitablefor the detection of the bar and pat genes in genetically modifed organisms, based on the intercalating dye SYBR Green I and theanalysis of the melting curves of the amplified products. Using this method, different amplification products specific for the barand pat genes were obtained and identified by their specific Tm. We have combined amplification of these products in a numberof duplex reactions and showed the suitability of the methods for identification of GMOs with a sensitivity of 0.9% in duplexreactions.